1. Field of the Invention
The present invention is directed to the prevention and treatment of an allergic disorder. In particular, the invention relates to the administration of polypeptides of the unique domain of the tyrosine kinase, Lyn, to the cells of a subject having, or at risk of having, an allergic disorder. The polypeptides act within the cells to bind the cytoplasmic domain of the high affinity receptor of IgE (Fcxcex5RI) and inhibit signaling through the receptor which would result in release of histamines and other substances associated with an allergic reaction, thereby preventing or treating an allergic disorder.
2. Background Art
The family of proteins known as the xe2x80x9cmultichain immune recognition receptorsxe2x80x9d includes the antigen receptors on B and T-lymphocytes and Fc receptors including the receptor with high affinity for IgE (Fcxcex5RI ) (1). Highly homologous in structure, all these receptors utilize, at least in part, a common mechanism to initiate cellular responses: multi-valent interactions with antigen leads to aggregation of the receptors and is followed by enhanced phosphorylation of tyrosines, in the Immune-Receptor Tyrosine-based Activation Motifs (ITAMs) within the cytoplasmic domains of the receptor itself, by a receptor-associated Src-family kinase (2).
Aggregation of the Fcxcex5RI on mast cells initiates a cascade of events leading to degranulation and release of mediators responsible for the symptoms of atopy. Among the earliest events in the Fcxcex5RI cascade is the phosphorylation of tyrosines in the ITAMs on the xcex2 and xcex3 chains of the receptor by the Src-family kinase, Lyn.
Several groups have studied the interaction between Fcxcex5RI and Lyn kinase by a variety of techniques and have demonstrated (7-10) a direct interaction between the kinase and the C-terminal cytoplasmic extension of the receptor""s xcex2 chain. Previous studies have also demonstrated that two forms of Lyn, designated Lyn A and Lyn B, are produced by differential mRNA splicing, both of which behave equivalently (5) and become equivalently attached to the receptor after chemical cross-linking (4).
For Fcxcex5RI, a xe2x80x9ctransphosphorylationxe2x80x9d mechanism has been demonstrated that accounts for the earliest cascade events (3,4). Data from these earlier studies showed that a small percentage of resting (unphosphorylated) receptors are constitutively associated with Lyn and this constitutive association with the kinase is an absolute requirement for the initial phosphorylation of the receptor.
The present invention provides polypeptides comprising the unique region of the Lyn kinase which bind the C-terminal cytoplasmic domain of the xcex2 chain of the Fcxcex5RI and inhibit activation of the Fcxcex5RI-mediated cascade of events that induce an allergic response and methods for the use of these polypeptides in treating or preventing allergic disorders.
The present invention provides an isolated polypeptide, in a pharmaceutically acceptable carrier, comprising a polypeptide having an amino acid sequence encoded by a nucleic acid which is at least 95% identical to a nucleic acid selected from the group consisting of: a) a nucleic acid encoding a polypeptide having the amino acid sequence of amino acids 1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:1; c) a nucleic acid encoding a polypeptide having amino acids 1-10 of the human tyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptide having amino acids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acid encoding a polypeptide having amino acids 27-66 of the human tyrosine kinase, Lyn A; and f) a nucleic acid encoding a polypeptide having any five or more contiguous amino acids of amino acids 1-66 of the human tyrosine kinase, Lyn A, wherein the polypeptide has substantially the same biologically functional activity of the polypeptide encoded by the nucleic acid sequence as set forth in (a), (b), (c), (d), (e) or (f).
Also provided is an isolated polypeptide, in a pharmaceutically acceptable carrier, comprising a polypeptide having an amino acid sequence encoded by a nucleic acid which is at least 95% identical to a nucleic acid selected from the group consisting of: a) a nucleic acid encoding a polypeptide having the amino acid sequence of amino acids 1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:2; and c) a nucleic acid encoding a polypeptide having any five or more amino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B, wherein the polypeptide has substantially the same biologically functional activity of the polypeptide encoded by the nucleic acid sequence as set forth in (a), (b) or (c).
Furthermore, the present invention provides an isolated polypeptide, which can be in a pharmaceutically acceptable carrier, produced from a cell transformed with a nucleic acid which is at least 95% identical to a nucleic acid selected from the group consisting of: a) a nucleic acid encoding a polypeptide having the amino acid sequence of amino acids 1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:1; c) a nucleic acid encoding a polypeptide having amino acids 1-10 of the human tyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptide having amino acids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acid encoding a polypeptide having amino acids 27-66 of the human tyrosine kinase, Lyn A; and f) a nucleic acid encoding a polypeptide having any five or more contiguous amino acids of amino acids 1-66 of the human tyrosine kinase, Lyn A, wherein the polypeptide has substantially the same biologically functional activity of the polypeptide encoded by the nucleic acid sequence as set forth in (a), (b), (c), (d), (e) or (f).
In addition, the present invention provides an isolated polypeptide, which can be in a pharmaceutically acceptable carrier, produced from a cell transformed with a nucleic acid which is at least 95% identical to a nucleic acid selected from the group consisting of: a) a nucleic acid encoding a polypeptide having the amino acid sequence of amino acids 1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:2; and c) a nucleic acid encoding a polypeptide having any five or more amino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B, wherein the polypeptide has substantially the same biologically functional activity of the polypeptide encoded by the nucleic acid sequence as set forth in (a), (b) or (c).
A method of treating or preventing an allergic disorder in a subject is also provided, comprising administering the nucleic acid of the present invention to a cell of the subject under conditions whereby the nucleic acid is expressed in the subject""s cells, thereby treating the allergic disorder.
The present invention also provides a fusion protein, comprising a polypeptide having an amino acid sequence encoded by a nucleic acid which is at least 95% identical to a nucleic acid selected from the group consisting of: a) a nucleic acid encoding a polypeptide having the amino acid sequence of amino acids 1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:1; c) a nucleic acid encoding a polypeptide having amino acids 1-10 of the human tyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptide having amino acids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acid encoding a polypeptide having amino acids 27-66 of the human tyrosine kinase, Lyn A; and f) a nucleic acid encoding a polypeptide having any five or more contiguous amino acids of amino acids 1-66 of the human tyrosine kinase, Lyn A, wherein the polypeptide has substantially the same biologically functional activity of the polypeptide encoded by the nucleic acid sequence as set forth in (a), (b), (c), (d), (e) or (f), and a ligand which binds to and is internalized by cells which express a high affinity receptor for IgE on the surface.
The present invention additionally provides a fusion protein, comprising a polypeptide having an amino acid sequence encoded by a nucleic acid which is at least 95% identical to a nucleic acid selected from the group consisting of: a) a nucleic acid encoding a polypeptide having the amino acid sequence of amino acids 1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:2; and c) a nucleic acid encoding a polypeptide having any five or more amino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B, wherein the polypeptide has substantially the same biologically functional activity of the polypeptide encoded by the nucleic acid sequence as set forth in (a), (b) or (c) and a ligand which binds to and is internalized by cells which express a high affinity receptor for IgE on the surface.
Further provided is a fusion protein, produced from a cell transformed with a nucleic acid which is at least 95% identical to a nucleic acid selected from the group consisting of: a) a nucleic acid encoding a polypeptide having the amino acid sequence of amino acids 1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:1; c) a nucleic acid encoding a polypeptide having amino acids 1-10 of the human tyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptide having amino acids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acid encoding a polypeptide having amino acids 27-66 of the human tyrosine kinase, Lyn A; and f) a nucleic acid encoding a polypeptide having any five or more contiguous amino acids of amino acids 1-66 of the human tyrosine kinase, Lyn A, wherein the polypeptide has substantially the same biologically functional activity of the polypeptide encoded by the nucleic acid sequence as set forth in (a), (b), (c), (d), (e) or (f), and encoding a ligand which binds to and is internalized by cells which express a high affinity receptor for IgE on the surface.
In addition, the present invention provides a fusion protein, produced from a cell transformed with a nucleic acid which is at least 95% identical to a nucleic acid selected from the group consisting of: a) a nucleic acid encoding a polypeptide having the amino acid sequence of amino acids 1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:2; and c) a nucleic acid encoding a polypeptide having any five or more amino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B, wherein the polypeptide has substantially the same biologically functional activity of the polypeptide encoded by the nucleic acid sequence as set forth in (a), (b) or (c), and encoding a ligand which binds to and is internalized by cells which express a high affinity receptor for IgE on the surface.
Finally, the present invention provides a method of treating or preventing an allergic disorder in a subject, comprising administering the fusion protein of the present invention to a cell of the subject, whereby the fusion protein treats or prevents the subject""s allergic disorder.
Various other objectives and advantages of the present invention will become apparent from the following detailed description.